Emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and NF-κB activation by disrupting lipid rafts in CD14-negative endothelial cells

BACKGROUND AND PURPOSE Emodin [1,3,8-trihydroxy-6-methylanthraquinone] has been reported to exhibit vascular anti-inflammatory properties. However, the corresponding mechanisms are not well understood. The present study was designed to explore the molecular target(s) of emodin in modifying lipopolysaccharide (LPS)-associated signal transduction pathways in endothelial cells. EXPERIMENTAL APPROACH Cultured primary human umbilical vein endothelial cells (HUVECs; passages 3-5) were pre-incubated with emodin (1-50 mg.mL(-1)). LPS-induced expression of pro-inflammatory cytokines [interleukin (IL)-1 beta, IL-6] and chemokines (IL-8; CCL2/MCP-1) were determined by reverse transcription-PCR and ELISA. Nuclear factor-kappa B (NF-kappa B) activation, inhibitor of kappa B (I kappa B) a degradation and Toll-like receptor-4 (TLR-4) were detected by immunocytochemistry and Western blotting. Cholesterol depletion [by methyl beta-cyclodextrin (MBCD), a specific cholesterol binding agent] and cholesterol replenishment were further used to investigate the roles of lipid rafts in activation of HUVECs. KEY RESULTS Emodin inhibited, concentration-dependently, the expression of LPS-induced pro-inflammatory cytokines (IL-1 beta, IL-6) and chemokines (IL-8, CCL2) and, in parallel, inhibited NF-kappa B activation and I kappa B alpha degradation in HUVECs. However, emodin did not inhibit the NF-kappa B activation and I kappa B alpha degradation induced by IL-1 beta. The cholesterol binding agent, MBCD, inhibited LPS-induced NF-kappa B activation in passaged HUVECs [which also lack the LPS receptor, membrane CD14 (mCD14)], showing that lipid rafts played a key role in LPS signalling in mCD14-negative HUVECs. Moreover, emodin disrupted the formation of lipid rafts in cell membranes by depleting cholesterol. CONCLUSIONS AND IMPLICATIONS Lipid rafts were crucial in facilitating inflammatory responses of mCD14-negative HUVECs to LPS. Emodin disrupted lipid rafts through depleting cholesterol and, consequently, inhibited inflammatory responses in endothelial cells.